This proposal is aimed at the study of the DNA sequence organization, evolution and structure of heterochromatin. One block of heterochromatin located at the base of the X chromosome of Drosophila melanogaster will be studied in detail since the bulk of its DNA can be isolated as a single satellite of density 1.688 g cm to the minus 3rd power. This satellite DNA consists largely of long tandem arrays of a 359 bp repeating unit occasionally interspersed with mobile genetic elements. By studying the arrangement of these mobile genetic elements within the D. melanogaster genome and the nucleotide sequence analysis of variant repeat units found in the satellite DNA we hope to determine the mechanism of evolution of these repetitive sequences. Moreover, by isolating and sequencing homologous DNAs from a number of closely related species (D. simulans, D. teissieri, D. orena, D. erecta, and D. mauritiana) we hope to prepare a complete evolutionary tree for this particular satellite DNA. We also propose to analyze the folding of the 1.688 satellite DNA into its highly folded heterochromatic state. We plan to use a competitive nitrocellulose filter binding assay to purify sequence specific DNA binding proteins. We will analyze the affinity, cooperativity, and degree of sequence specificity of such proteins. We will utilize a crude chromatin assembly system isolated from Drosophila embryos to analyze the interactions of sequence specific proteins with histones and to study the arrangementss of these proteins with nucleosomes, as well as any other components which associate with the satellite chromatin in vitro. By studying the interactions between both the sequence specific and on-sequence specific proteins we hope to understand not only the nature of the molecular forces that both fold long tandem arrays into a condensed form, but gain insight into interchromosomal homologous associations such as ectopic pairing, meiotic pairing chromocentral aggregation, and polytene chromosome formation.